Microcapillary network based scaffold

ABSTRACT

A scaffold is provided, the scaffold comprising: at least one inlet tube; at least one outlet tube; and a plurality of porous elongated microtubes, wherein each one of said porous elongated microtube has an inner diameter of 5-100 micrometers, wherein said plurality of elongated microtubes extend from said at least one inlet tube to said at least one outlet tube and is in fluid communication thereto, Further provided is a method for producing and using the scaffold, such a s for tissue engineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 15/759,940 filedMar. 14, 2018, which is a National Phase of PCT Patent Application No.PCT/IL2016/051032 having International filing date of Sep. 18, 2016,which claims the benefit of priority U.S. Provisional Patent ApplicationNo. 62/220,129 filed on Sep. 17, 2015 entitled MICROCAPILLARY NETWORKBASED SCAFFOLD. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD OF INVENTION

The present invention relates to the field of tissue engineering, andmore particularly to the use of synthetic scaffold for the preparationof prosthetic implants.

BACKGROUND OF THE INVENTION

Tissue engineering techniques generally require the use of a scaffold asa three-dimensional template for initial cell attachment and subsequenttissue formation. As such, scaffold design is one of the most importantaspects of tissue engineering. Appropriate selection of a scaffold is akey factor in the process of producing a viable and clinically relevantengineered tissue.

A scaffold is expected to possess the following characteristics for usein tissue engineering: (i) a three-dimensional porous structure thatallows cell/tissue growth and flow transport of nutrients and metabolicwaste; (ii) biodegradable or bioresorbable with a controllabledegradation and resorption rate to match cell/tissue growth in vitroand/or in vivo; (iii) conducive surface chemistry for cell attachment,proliferation, and differentiation; (iv) mechanical properties to matchthose of the tissues at the site of implantation; and (v) processability to form a variety of shapes and sizes for various applications.

In addition, in order to improve the survival and function of theimplanted engineered tissue, adequate vascularization within thescaffold is crucial. Tissue vascularization is essential for delivery ofnutrients (amino acids, glucose and oxygen) to the tissue and clearanceof the metabolism byproducts from the tissue. Numerous techniques fordevelopment of vascularized tissue have recently emerged and areclassified into two major categories: (a) vasculogenesis andangiogenesis-based techniques and (b) prevascularization-basedtechniques. The former are characterized by the ingrowth of newly formedblood vessels from the host microvasculature into the implantedengineered tissue and includes (1) micropatterning of vascularmorphogenesis, (2) use of functionalized biomaterials to promotevasculogenisis and angiogenesis, (3) use of growth factor gradients and(4) co-culture of multiple cell types and control of cell-cellinteractions. These techniques can be utilized to promote the formationof vascular networks in 3D engineered constructs in a regulated manner,but their central drawback lies in the time-consuming process ofpromoting vasculogenesis and angiogenesis, at a critical time whensurvival rates of implanted scaffolds are determined. Theprevascularization-based techniques are founded on generating ofpreformed microvascular networks within tissue constructs prior to theirimplantation, which later further develop and interconnect with hostblood vessels at the implantation site. The key advantage of thesetechniques is the capacity for immediate blood perfusion within theconstructs upon implantation, which boosts the proliferation and growthof the cells. However, despite these advanced techniques, clinical useof engineered tissues and tissue substitutes is still largely restrictedto avascular or thin tissues, and the marked progress achieved insmall-scale tissue engineering applications in vitro was ultimatelystalled due to the lack of vascular perfusion when scaled up to a sizesrelevant for implantation and disease treatment.

Therefore, engineering a complex bulk tissue that can maintain itsviability in vivo by transporting essential growth factors throughoutthe entire volume of the scaffold, remains an unmet need in the field oftissue engineering in general, and bone tissue engineering inparticular.

SUMMARY OF THE INVENTION

According to a first aspect the invention provides a scaffoldcomprising:

at least one inlet tube;

at least one outlet tube; and

a plurality of porous elongated microtubes, wherein each one of saidporous elongated microtube has an inner diameter of 5-100 micrometers,

-   -   wherein said plurality of elongated microtubes extend from said        at least one inlet tube to said at least one outlet tube and is        in fluid communication thereto.

In some embodiments, the scaffold further comprises a plurality offibers having a diameter range of 0.5-10 micrometers. In someembodiments, the plurality of fibers is dispersed upon a portion of eachof said plurality of porous elongated microtubes.

In some embodiments, the scaffold further comprises a plurality ofbioactive particles embedded in between said plurality of fibers. Insome embodiments, the scaffold further comprises a plurality ofbioactive particles embedded in between said plurality of fibers and atleast a portion of said porous elongated microtubes. In someembodiments, the bioactive particles have a range of 200-1500micrometers in diameter.

In some embodiments, the plurality of bioactive particles comprises oneor more type of osteoconductive particles. In some embodiments, the oneor more types of the osteoconductive particles are selected from thegroup consisting of: calcium carbonate, hydroxyapatite (HA),demineralized bone material, morselized bone graft, cortical cancellousallograft, cortical cancellous autograft, cortical cancellous xenograft,tricalcium phosphate, corraline mineral and calcium sulfate. In someembodiments, the particles comprise hydroxylapatite (HA) and calciumcarbonate.

In some embodiments, at least a portion of the scaffold is printed,molded, casted, polymerized, or electrospun. In some embodiments, atleast one of said inlet tube, said outlet tube and said porous elongatedmicrotubes are electrospun tubes. In some embodiments, said plurality offibers are electrospun fibers. In some embodiments, the electrospuntubes or fibers comprise a polymer (or are formed from a polymericsolution) selected from the group consisting of: biodegradable polymers,non-biodegradable polymers and a combination thereof. In someembodiments, the polymer is selected from the group consisting of:polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA),and poly(Lactide-co-Glycolide) (PLGA), poly(orthoester), apoly(phosphazene), poly(or polycaprolactone, polyamide, polysaccharide,albumine and collagen.

In some embodiments, the inlet tube and the outlet tube have,independently, an inner diameter of range of 2,000-10,000 micrometers.

In some embodiments, the inlet tube and the outlet tube have,independently, a wall thickness range of 50-2,000 micrometers.

In some embodiments, the plurality of porous elongated microtubes have awall thickness range of 0.5-50 micrometers.

In some embodiments, an average diameter of a pore of the plurality ofporous elongated microtubes is 0.1-5 micrometers.

In some embodiments, the scaffold further comprises at least one agentfor promoting cell adhesion, colonization, proliferation and/ordifferentiation. In some embodiments, the scaffold further comprises atleast one agent for promoting cell adhesion selected from the groupconsisting of: gelatin, fibrin, fibronectin and collagen.

In some embodiments, the scaffold further comprises a plurality ofcells. In some embodiments, the scaffold further comprises a pluralityof cells seeded on and/or within the plurality of fibers. In someembodiments, the scaffold is adapted for cellular growth.

In some embodiments, the plurality of cells is selected from the groupconsisting of: adipose-derived stem cells, mesenchymal cells,mesenchymal stem cells, vascular smooth muscle cells, adipogenic cells,osteoprogenitors cells, osteoblasts, osteocytes, chondroblasts,chondrocytes and osteoclasts, endothelial progenitor cells,hematopoietic progenitor cells, micro vascular endothelial cells andmacro vascular endothelial cells, beta cells, hepatocytes and acombination thereof

According to another aspect, the invention provides a method ofproducing a tissue, the method comprising:

providing a scaffold comprising:

-   -   at least one inlet tube;    -   at least one outlet tube; and    -   a plurality of porous elongated microtubes, wherein each one of        said porous elongated microtube has an inner diameter of 5-100        micrometers,    -   wherein said plurality of elongated microtubes extend from said        at least one inlet tube to said at least one outlet tube and is        in fluid communication thereto;    -   seeding cells on said plurality of porous elongated microtubes        of said scaffold; and    -   providing fluid (e.g., liquid) containing nutrients through said        inlet of said scaffold, so as to provide nutrients from pores of        said plurality of porous elongated microtubes to said cells;

thereby producing said tissue.

In some embodiments, the scaffold further comprises a plurality offibers dispersed upon each of said plurality of porous elongatedmicrotubes. In some embodiments, the cells are seeded on and/or withinsaid plurality of fibers.

In some embodiments, the tissue is suitable for being implanted into asubject in need thereof.

In some embodiments, the inlet and the outlet of the scaffold issuitable for being surgically connected to a vascular system of asubject in need thereof, thereby providing fluid communication betweenthe subject's vascular system and said scaffold.

In some embodiments, the cells are selected from the group consistingof: adipose-derived stem cells, mesenchymal cells, mesenchymal stemcells, vascular smooth muscle cells, adipogenic cells, osteoprogenitorscells, osteoblasts, osteocytes, chondroblasts, chondrocytes andosteoclasts, endothelial progenitor cells, hematopoietic progenitorcells, micro vascular endothelial cells and macro vascular endothelialcells, beta cells, hepatocytes and a combination thereof.

In some embodiments of the disclosed method, the scaffold furthercomprises plurality of bioactive particles embedded in between saidplurality of fibers.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thefigures and by study of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are schematic presentations of the microcapillary basedscaffold;

FIGS. 2A-D are optical images of (A) an electrospun capillary, (B-D) PCLelectrospun fibers (9 wt. %).

FIG. 3 is a schematic illustration of a permeation testing setup of anelectrospun tube connected to inlet/outlet;

FIGS. 4A-D present images of the complex scaffold: (A) the complexscaffold: Pro-Osteons dispersed on and in between the PCL-electrospuntubes. The indicated region is the region that is seeded with cells, (B)the vasculature-like system: made of PCL-electrospun tubes, (C-D) across-sectional view of the vasculature-embedded scaffold, showing threePCL tubes surrounded with layers of Pro-Osteon particles and PCL fiber;

FIGS. 5A-C present SEM images of the complex scaffold: (A) cross sectionof the complex scaffold showing the PCL fibers and the Pro-Osteonparticles dispersed on and in between the PCL-electrospun tubes, (B) thescaffold bulk including Pro-Osteon particles and electrospun PCL fibers,(C) electrospun PCL tube (different magnifications (×50, ×40 & ×150) areshown);

FIG. 6 shows the complex scaffold connected to a medium-flow bioreactor;

FIGS. 7A-C present gel electrophoresis results: (A) the proteins thatpassed through the PCL tubes walls and into the PBS bath, (B)quantification of the protein bands observed on the gel, (C) increase ofprotein concentrations in the inner PBS bath;

FIG. 8 is a graph showing cells proliferation when cultured on complexscaffolds under static conditions. The Alamar blue-based viability assayshowed increase of proliferation rates of MSCs in correlation to theculture days. Error bars are the standard deviation (SD) collected fromthree samples;

FIGS. 9A-D show H&E-stained histological sections of the MSC-seededcomplex scaffolds. Complex scaffolds were seeded with MSCs, and culturedunder static conditions for one week and then in dynamic conditions foran additional week before being fixed and stained. Differentmagnifications are shown: (A, C) ×4, (B, D) ×10. The PCL tubes areindicated by asterisks.

FIGS. 10A-C show SEM images of the MSC-seeded complex scaffold grownunder dynamic conditions: (A) cross-section of a PCL tube showing theintegration of cells along the tube walls, (B-C) cells were distributedon and in between the PCL fibers and Pro-Osteon particles (differentmagnifications (×450, ×2680 and ×415) are shown); and

FIGS. 11A-I show H&E- and Trichrome-stained histological sections of theimplanted MSC-embedded complex scaffold in animal models. Cell-seededscaffolds were implanted in an ectopic animal model in order to examinetheir biocompatibility. Eight weeks later, the implanted scaffolds wereextracted and histological sections were prepared and stained with H&Estaining and Trichrome staining. PCL tubes, PCL fibers and Pro-Osteonparticles are indicated by black asterisks, arrows and crosses,respectively. Blood vessels, muscle tissue and collagen accumulation arenoted by arrows, different magnifications are shown: (A, C, E, F and H)×4, (B, D, G and I) ×10;

FIGS. 12A-B demonstrate a Hollow Fiber Reactor (HFR) system FIG. 12A isa photograph of a Hollow Fiber Reactor (HFR) system; and FIG. 12B is aschematic illustration of the Hollow Fiber Reactor (HFR) system (1280)of FIG. 12A;

FIGS. 13A-B show photographs following Giemsa staining of Coralparticles from the inner side (A) and the outer side (B) of the HFRsystem;

FIGS. 14A-B show photographs following Hematoxylin& Eosin staining ofHFR construct after one week of cultivation, magnification ×10, thesephotographs (A and B) demonstrate that the cells between the mineralparticles were embedded within the PCL fibers, and generated organizedconnective tissue around and between the mineral particle;

FIGS. 15A-B show photograph following MTT staining of Mineral particleswith live cells (A), and mineral particles without cells used as control(B);

FIGS. 16A-B are graphs showing FACS results for the osteogenic markerALP from MSCs grown in a Static HFR system (A) and dynamic HFR system(B).

FIGS. 17A-D are bar graphs showing Real-Time PCR results of DLX5expression at day 0 and day 9 in MSCs grown in a static system (A) or adynamic system (B), and SP7 expression at day 0 and day 9 in MSCs grownin a static system (C) or a dynamic system (D);

FIGS. 18A-B are photographs of Giemsa staining (A) and AC-LDL staining(B) of human adipose microvascular endothelial cells (HAMEC) grown inmicrocapillary reactor system;

FIGS. 19A-B are a photograph (A) and a schematic illustration (B) of amicrocapillary system for testing permeability to human plasma proteins;

FIGS. 20A-C are bar graphs demonstrating concentrations of human serumalbumin (A), IgG (B) and lysozyme (C) during the plasma circulation intothe microcapillary construct;

FIGS. 21A-H are photographs demonstrating “End to End” anastomosis ofvasculature-like system in SD rat using microsurgery technique: FIG. 21Ashows exposure of femoral artery and vain before the microsurgeryprocedure, FIG. 21B shows “End to End” anastomosis of the microcapillarygraft to the artery and vain vessels, FIG. 21C is a magnification ofFIG. 21B, FIG. 21D is a magnification of FIG. 21C with focus on theconnected microcapillary graft, FIG. 21E shows the microcapillary graftfollowing the “end to end” anastomosis while the microcapillary graft isalready connected and enables the blood circulation, FIG. 21F is amagnification of FIG. 21E, and FIG. 21G is a magnification of FIG. 21F,both focused on the “end to end” anastomosis area, and FIG. 21H showsthe whole animal following stitching and closure of the surgery area;

FIGS. 22A-B are photographs demonstrating the measurement of blood flowfrom artery to vein in the anastomosis site (A) and the measured valuefor blood flow in the artery as displayed on the laser flow meter (B);

FIGS. 23A-E are photographs demonstrating opening of the stitches (A)and exposure of the anastomosis site (B) performed one-day posttransplantation to allow measurement of blood flow from artery to veinby laser Doppler (C), and the measured value for blood flow in theartery (D) and vein (E) as displayed on the laser flow meters;

FIG. 24 vasculature-like system extracted 14 days following thetransplantation.

FIG. 25A-D are histological images magnified by×X10 (A) ×40 (B) ×60 (C)and ×100 (D) of the transplanted vasculature-like system extracted fromthe rats 14 days after the transplantation and stained by Hematoxylin &Eosin staining;

FIGS. 26A-D are photographs demonstrating the anastomosis ofheparin-soaked scaffold to the femoral artery from one side (A) and thescaffold connected to femoral artery from one side and the femoral veinfrom the other side with no apparent leakage from the connection sites(B), FIGS. 26C-D are photographs demonstrating the measurement of bloodflow through the transplant in anesthetized rats (C) and the measuredvalue as displayed on the laser flow meter (D);

FIG. 27 is a graph plotting flow rate (milliliters/second) versus pumprate (RPM); and

FIG. 28 is a bar graph showing fluid loss from the scaffold (presentedas % of fluid flow) in capillary like tubes of human (diameter of 0.5mm, width of 237 micrometers) and vein like tubes of rats (diameter of1.5 mm, width of 390 micrometers) under a fluid flow rate of 0.005cm³/sec inside the scaffold compared to a fluid flow rate of 0.038cm³/sec inside the scaffold.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments, provides a 3-dimensional(3D) scaffold that supports growth of cells in a tissue construct, thescaffold comprises a porous tubular system that serves as a built-invascular system. In some embodiments, the invention provides a scaffoldcomprising at least one inlet tube; at least one outlet tube; and aplurality of porous elongated microtubes, wherein each one of saidporous elongated microtube has an inner diameter of 5-100 micrometers,and wherein said plurality of elongated microtubes extend from said atleast one inlet tube to said at least one outlet tube and is in fluidcommunication thereto.

In some embodiments, the scaffold provides therein a tubular systemfacilitating transport of nutrients, gases and metabolites from liquidflux within the tubular system through the pores to cells attachedthereto. In some embodiments, the scaffold's tubular system facilitatestransport of by-products of metabolites from the cells.

The present invention is based in part on the finding that the scaffoldof the invention serves as an adequate extracellular matrix (ECM) forcell integration, attachment, proliferation, growth and differentiationas a result of its structure.

In embodiments wherein the designated tissue is a bone, the scaffold maybear features required for effective bone-tissue engineered constructsand is characterized by osteoconductiveness that induces mesenchymalstem cells (MSC) differentiation into bone-forming cells. In someembodiments, the scaffold can serve as a bioreactor system providingappropriate growth conditions for MSC proliferation and differentiationinto bone-forming cells in vitro and for their subsequent developmentinto bone structure in vivo after anastomosis with host blood vessels.

Reference is now made to FIG. 1A, which is a schematic illustration of ascaffold 100 according to an embodiment of the invention. Scaffold 100comprises an inlet tube 102 which is fluidly-connected to plurality ofporous elongated microtubes 104 which extend in fluid communication toan outlet tube 106. Optionally, inlet tube 102 may consist of pluralityof inlet tubes such as inlet tubes 102 a, 102 b, 102 c. Optionallyoutlet tube 106 may consist of plurality of inlet tubes such as inlettubes 106 a, 106 b, 106 c. Reference is now made to FIG. 1B, which is anenlarged view of a section of porous elongated microtubes 104. Aplurality of fibers 108 that may serve as a release system ofangiogenic/growth factors are dispersed upon porous elongated microtubes104. Optionally, bioactive particles 110 (e.g., osteoconductiveparticles) may be embedded within plurality of fibers 108. Optionally, aplurality of cells 112 may be seeded within plurality of fibers 108. Fora non-limiting example, plurality of cells may include bone formingcells. The wide arrow depicts the flow of blood within porous elongatedmicrotubes 104 and narrow arrows depict the flow of nutrients and oxygen(O₂) from porous elongated microtubes 104 to plurality of cells 112.FIGS. 1C-D illustrates forming of cell tissue upon porous elongatedmicrotubes 104.

Scaffold

According to another aspect, the present invention provides a scaffoldcomprising: at least one inlet tube; at least one outlet tube; and aplurality of porous elongated microtubes, wherein each one of saidporous elongated microtube has an inner diameter of 5-100 micrometers,and wherein said plurality of elongated microtubes extend from said atleast one inlet tube to said at least one outlet tube and is in fluidcommunication thereto.

In some embodiments, the diameters of the inlet and/or outlet tubes is2-1000, or 200-1000, or 2-100 folds greater than the diameter of theporous elongated microtube. In some embodiments, the inlet tube and theoutlet tube have an inner diameter range of 2,000-10,000 micrometers. Insome embodiments the inlet tube and the outlet tube have a wallthickness range of 50-2,000 micrometers. In some embodiments theplurality of porous elongated microtubes has a wall thickness range of0.5-50 micrometers. In some embodiments the ratio between the number ofporous elongated microtubes and any one of the inlet and outlet tubes isin the range of 1:1-10:1, or alternatively 1:1-5:1, or alternatively1:1-3:1, or alternatively 1:1-50:1, or alternatively 1:1-100:1.

The term “porous” as used herein relates to a plurality of openings,pores, or holes that may be filled (permeated) by water, air or othermaterials. In some embodiments, pores are not permeable to cells such asmammalian cells. In some embodiments, a diameter of a pore is less than10 micrometers. In some embodiments, a diameter of a pore is between0.1-5 micrometers. In some embodiments, a diameter of a pore is between0.1-10 micrometers, between 0.5-5 micrometers, or alternatively between1-10 micrometers.

As used herein throughout, the term “fluid communication” meansfluidically interconnected, and refers to the existence of a continuouscoherent flow path from one of the components of the system to the otherif there is, or can be established, liquid and/or gas flow through andbetween the ports, when desired, to impede fluid flow therebetween.

In some embodiments, the scaffold further comprises a plurality offibers having a diameter range of 0.5-10 micrometers, or 0.1-20micrometers, or 1-5 micrometers. In some embodiments, the fibers, or atleast some of the fibers may be hollow. In some embodiments, a distancebetween adjacent fibers ranges between 20 micrometers and 300micrometers. In some embodiments the fibers are arranged in a mesh. Insome embodiments, the mesh comprises openings defined between adjacentfibers. In some embodiments, the openings have a diameter range of 20micrometers and 300 micrometers. In some embodiments the plurality offibers and/or the mesh is dispersed upon at least a portion of thescaffold. In some embodiments, the plurality of fibers and/or the meshis dispersed upon a portion of each of said plurality of porouselongated microtubes.

The term “scaffold” as used herein refers to a porous, artificial,three-dimensional structure comprising biocompatible material thatprovides a surface suitable for adherence and proliferation of cells.Biocompatible, as used herein, is intended to describe materials that,are non-toxic to cells in vitro and upon administration in vivo, do notinduce undesirable long-term effects. As used herein the term “in vitro”refers to any process that occurs outside a living organism. As usedherein the term “in-vivo” refers to any process that occurs inside aliving organism.

As used herein, the term “diameter” refers to the largest lineardistance between two points on the surface of a described element (e.g.,tube, fiber, openings). The term “diameter”, as used herein, encompassesdiameters of spherical elements as well as of non-spherical elements.

In some embodiment, the scaffold is biodegradable. Biodegradable, asused herein, is intended to describe materials that are biologicallydegraded in vivo.

Scaffold of the present invention, or a portion thereof may be printed,molded, casted, polymerized, or electrospun.

In some embodiments, the scaffold contains or consists of electrospunmaterial (e.g. macro micro or nanofibers).

In some embodiments the scaffold may consists of, or include, one ormore polymers selected from the group consisting of: biodegradablepolymers and non-biodegradable polymers. In some embodiments thescaffold may consists of or include any of the following materials:polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA),poly(Lactide-co-Glycolide) (PLGA), poly(orthoester), apoly(phosphazene), a polyamide, a polysaccharide, albumin, collagen(e.g., collagen I or IV), fibrin, hyaluronic acid, poly(vinyl alcohol)(PVA), Polyhydroxybutyrate (PHB), poly(ethylene oxide) (PEO), fibrin,polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol(PEG), alginate, chitosan copolymers or mixtures thereof.

In some embodiments, the scaffold further comprises bioactive agents. Asused herein, the terms “bioactive” is used to refer to any effect on,interaction with, or response from living cells and/or tissue. The term“bioactive agent” refers to a molecule that exerts an effect on a cellor tissue.

Representative examples of types of bioactive agents includetherapeutics, vitamins, electrolytes, amino acids, peptides,polypeptides, proteins, carbohydrates, lipids, polysaccharides, nucleicacids, nucleotides, polynucleotides, glycoproteins, lipoproteins,glycolipids, glycosaminoglycans, proteoglycans, growth factors,differentiation factors, hormones, neurotransmitters, prostaglandins,immunoglobulins, cytokines, and antigens. Various combinations of thesemolecules can be used. Examples of cytokines include macrophage derivedchemokines, macrophage inflammatory proteins, interleukins, tumornecrosis factors. Examples of proteins include fibrous proteins (e.g.,collagen, elastin) and adhesion proteins (e.g., actin, fibrin,fibrinogen, fibronectin, vitronectin, laminin, cadherins, selectins,intracellular adhesion molecules, and integrins). In various cases, thebioactive agent may be selected from fibronectin, laminin,thrombospondin, tenascin C, leptin, leukemia inhibitory factors, RGDpeptides, anti-TNFs, endostatin, angiostatin, thrombospondin, osteogenicprotein-1, bone morphogenic proteins, osteonectin, somatomedin-likepeptide, osteocalcin, interferons, and interleukins. In someembodiments, the bioactive agent includes a growth factor,differentiation factor, or a combination thereof

As used herein, the term “growth factor” refers to a bioactive agentthat promotes the proliferation of a cell or tissue. Representativeexamples of growth factors that may be useful include transforminggrowth factor-α (TGF-α), transforming growth factor-β (TGF-β),platelet-derived growth factors (PDGF), fibroblast growth factors (FGF),nerve growth factors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF andneurotrophins, brain derived neurotrophic factor, cartilage derivedfactor, bone growth factors (BGF), basic fibroblast growth factor,insulin-like growth factor (IGF), vascular endothelial growth factor(VEGF), EG-VEGF, VEGF-related protein, Bv8, VEGF-E, granulocyte colonystimulating factor (G-CSF), insulin like growth factor (IGF) I and II,hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stemcell factor (SCF), keratinocyte growth factor (KGF), transforming growthfactors (TGF), (e.g., TGFs α, β, β1, β2, and β3), any of the bonemorphogenic proteins, skeletal growth factor, bone matrix derived growthfactors, and bone derived growth factors and mixtures thereof.

As used herein the term “differentiation factor” refers to a bioactiveagent that promotes the differentiation of cells. Representativeexamples include neurotrophins, colony stimulating factors (CSF), andtransforming growth factors. Some growth factors may also promotedifferentiation of a cell or tissue. Some differentiation factors alsomay promote the growth of a cell or tissue. For example, TGF may promotegrowth and/or differentiation of cells. In some embodiments, thescaffold comprises at least one bioactive agent for promoting celladhesion, colonization, proliferation and/or differentiation. In someembodiments, the at least one agent for promoting cell adhesion isselected from the group consisting of: gelatin, fibrin, fibronectin andcollagen.

The bioactive agent may be incorporated into the scaffold in a varietyof different ways. In one embodiment, the bioactive agent is locatedand/or formulated for controlled release to affect the cells or tissuesin or around the oriented nanofiber structures. For instance, it may bedispersed in a controlled release matrix material. In one embodiment,the bioactive agent is provided in lipid microtubules or nanoparticlesselected to modulate the release kinetics of the bioactive agent. Suchparticles may be dispersed among the nanofibers, or provided within thescaffold. In another embodiment, the bioactive agent is actuallyintegrated into, forms part of, the tubes, microtubes and/or fibersthemselves. This may be done, for example, by adding the bioactive agentto a polymer solution prior to electrospinning the solution to form thetubes, microtubes and/or fibers themselves. Release of the bioactiveagent may be controlled, at least in part, by selection of the type andamounts of biodegradable matrix materials in the nanoparticles ornanofibers.

In some embodiments the scaffold further comprises cells such asendothelial cells attached thereto within one or more the tubes ormicrotubes of the scaffold. One skilled in the art will appreciate thatincorporation of endothelial cells within tubes of the scaffold mayenhance vascular tissue formation, e.g., so as to replace the scaffold'ssynthetic vascular tubes/microtubes which are degraded in vivo.

In some embodiments, the scaffold further comprises plurality ofbioactive particles having a range of 200-1500 micrometers in diameter.In some embodiments, particles of the invention are larger than 1micrometer in diameter. Particles of the invention may have any shape orform (e.g., spherical, triangular, rectangular, etc.). In someembodiments, the particles are embedded within the scaffold. In someembodiments, the particles are embedded in between the plurality offibers dispersed upon the scaffold or a portion thereof

In some embodiments, the bioactive particles are ceramic particles. Asused herein, the term “ceramic” is intended to refer to an inorganic,nonmetallic material, typically crystalline in nature, though it couldbe amorphous as well. Ceramics generally may be compounds formed betweenmetallic and nonmetallic elements, such as, for example, aluminum andoxygen (e.g., alumina—Al₂O₃), calcium and oxygen (e.g., calcia—CaO),silicon and oxygen (e.g., silica—SiO₂) and other analogous oxides,nitrides, borides, sulfides, and carbides as well as carbon matrices.However, “ceramic”, as used herein, should not be unduly construed asbeing limited to a ceramic body in the classical sense, that is, in thesense that it consists entirely of inorganic materials, but ratherrefers to a body which is predominantly ceramic with respect to eithercomposition or dominant properties.

In some embodiments, the plurality of particles comprises one or moretype of osteoconductive particles. As used in here “osteoconductive”refers to the ability of a substance to serve as a suitable template orsubstance along which bone may grow. In one embodiment, the one or moretypes of the osteoconductive particles are osteoconductive ceramicparticles selected from the group consisting of: calcium carbonate,hydroxyapatite (HA), demineralized bone material, morselized bone graft,cortical cancellous allograft, cortical cancellous autograft, corticalcancellous xenograft, tricalcium phosphate, coralline mineral andcalcium sulfate. In some embodiments, the bioactive particles comprisehydroxylapatite (HA) and calcium carbonate.

Applications

A scaffold according to the present invention can be used for a widevariety of applications. Embodiments of the scaffolds disclosed hereinare suitable for uses such as for cell culture and cell transplantation.In some embodiments the scaffold may serve as a bioreactor systemproviding appropriate growth conditions for cells in vitro. In someembodiments, the scaffold may serve as perfusion bioreactor thatprovides for immediate supply of nutrients and gases to cells grown in atissue culture.

Reference is now made to FIG. 12B which is a schematic illustration of ahollow fiber reactor (HFR) system 1270 comprising a scaffold (alsoreferred to as microcapillary system) 1273, according to an embodimentof the invention. Scaffold 1273 is connected to an inlet tube 1275 andan outlet tube 1276. Optionally, inlet tube may be equipped with aperistaltic pump 1277 to facilitate constant circulation of a growthmedium. Optionally, the circulation of growth medium facilitates aturbulence movement of the growth medium. Scaffold 1273 is enclosedwithin a HFR vessel 1274. The HFR system may further include a mediumreservoir 1271. Optionally, medium reservoir 1271 is equipped with a cap1272. Optionally, inlet tube 1275 and outlet tube 1276 which areconnected to scaffold 1270 contact medium reservoir 1271 via ports (notshown) in cap 1272. Optionally, waste removal is done using a wasteoutlet 1279 via a port (not shown) in cap 1272. Optionally, growthmedium replenishment is done using a feed inlet 1280 via a port (notshown) in cap 1272. Optionally, system 1270 is continuously aeratedthrough aeration inlet 1281 with filtered air/CO₂ (95%/5%, respectively)gas mixture bubbling out through a sparger 1282 dipped into the growthmedium. Optionally, pressure is release through an output 1283.Optionally, cell seeding is conducted through an HFR inoculation port1278. Following cell seeding onto scaffold 1273, inoculation port 1278is closed and during closed system is maintained during the 3D growthphase of the cells. In some embodiment, the flow rate of the growthmedium is regulated. In some embodiments, the required flow rate ofgrowth medium is determined according to the characteristics of thescaffold (e.g. diameter). In some embodiment, the RPM of pump 1277 motoris used as the main control of the flow rate. For a non-limitingexample, when using a microcapillary system including a capillary tube,which has a diameter of 0.5 mm, connected at each side to a one of twovein like tubes having a diameter of 0.86 mm the required RPM is between10 and 50. As exemplified in example 10 the required flow rates and RPMmay be calculated (i.e. by applying equations 2-4) for specificapplications.

The term “bioreactor” as used herein means any apparatus, which providesbiologically active, protected environment suitable for cultivation ofcells. The term “perfusion bioreactor” as used herein means afluidized-bed reactor for cell culture designed for continuous operationas a perfusion system, i.e., a system in which fresh medium is fed tothe bioreactor at the same rate as spent medium is removed. In someembodiments, the scaffold is implantable, and may be surgicallyconnected to a subject's blood vessels. In some embodiments the scaffoldmay serve as a bioreactor system providing appropriate growth conditionsfor cells in vitro and for their subsequent development into bonestructure in vivo after anastomosis with host blood vessels. As usedherein, the term “anastomosis” refers to the joining together of twohollow structures, for a non-limiting example, two arteries or veins, toachieve continuity. An anastomosis can be end-to-end, side-to-side, orend-to-side depending on the circumstances of the requiredreconstruction or bypass procedure.

In some embodiments, the scaffold is used to produce a tissue. In someembodiments the method for producing a tissue comprises: providing thescaffold of the invention, seeding cells on said plurality of porouselongated microtubes of said scaffold; and providing liquid containingnutrients through said inlet of said scaffold, so as to providenutrients from pores of said plurality of porous elongated microtubes tosaid cells; thereby producing the tissue.

In some embodiments, the method comprises a preliminary step ofdetermining a desired flow rate of said liquid containing nutrientsthrough said scaffold. In some embodiments, the desired flow rate issuitable for producing a tissue. In some embodiments, the desired flowrate is suitable for the step of cells seeding. In some embodiments, thedesired flow rate is suitable for the step of cells culturing. In someembodiments, the flow rate is controlled by a pump. In some embodiments,an RPM range of the pump is determined according to a desired flow rate.

The term “subject” as used herein refers to an animal, more particularlyto non-human mammals and human organism. Non-human animal subjects mayalso include prenatal forms of animals, such as, e.g., embryos orfetuses. Non-limiting examples of non-human animals include: horse, cow,camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit,hamster, guinea pig, pig. In one embodiment, the subject is a human.Human subjects may also include fetuses. In one embodiment, a subject inneed thereof is a subject afflicted with a fractured bone, a boneinjury, diminished bone mass and/or bone abnormality.

The terms “liquid”, “fluid” and “media” as used interchangeably hereinrefer to water or a solution based primarily on water such as phosphatebuffered saline (PBS), or water containing a salt dissolved therein. Theterm aqueous medium can also refer to a cell culture medium. The term“cell culture medium” refers to any liquid medium which enables cellsproliferation. Growth media are known in the art and can be selecteddepending of the type of cell to be grown. For example, a growth mediumfor use in growing mammalian cells is Dulbecco's Modified Eagle Medium(DMEM) which can be supplemented with heat inactivated fetal bovineserum.

The term “nutrients” may include but is not limited to fats, glucose,mono- or oligo-saccharides, minerals, trace elements and/or vitamins.Nutrients may further include one or more gaseous components such asprimarily oxygen and carbon dioxide. Nutrients may further include oneor more metabolite.

The term “metabolite” or “metabolites” as used herein designatescompounds that are naturally produced by an organism (such as a plant oranimal) and that are directly involved in the normal growth, developmentor reproduction of the organism. This includes, but is not limited to,any compound produced by plant or animal cells, or genetically modifiedplant or animal cells, such as proteins or other types of chemicalcompounds.

The terms “cell” and “cells” as used herein, refer to isolated cells,cell lines (including cells engineered in vitro), any preparation ofliving tissue, including primary tissue explants and preparationsthereof. Any type of cell can be added to the scaffold for culturing andpossible implantation, including cells of the muscular and skeletalsystems, such as chondrocytes, fibroblasts, muscle cells and osteocytes,parenchymal cells such as hepatocytes, pancreatic cells (including Isletcells), cells of intestinal origin, and other cells such as nerve cellsand skin cells, either as obtained from donors, from established cellculture lines, or even before or after genetic engineering. Pieces oftissue can also be used, which may provide a number of different celltypes in the same structure. The scaffold can also be used as athree-dimensional in vitro culture system for attachment-dependentcells, e.g., hepatocytes in a 3D micro environment which mimics thephysiological micro environment more closely. In some embodiments, cellsare selected from the group consisting of: adipose-derived stem cells,mesenchymal cells, mesenchymal stem cells, vascular smooth muscle cells,adipogenic cells, osteoprogenitors cells, osteoblasts, osteocytes,chondroblasts, chondrocytes and osteoclasts, endothelial progenitorcells, hematopoietic progenitor cells and a combination thereof.

Scaffolds or portions thereof described herein can be used to generatesynthetic organs or tissues or portions thereof, including but notlimited to, respiratory tissues (e.g., tracheal, bronchial, esophageal,alveolar, and other pulmonary or respiratory tissues), circulatorytissues (e.g., arterial, venous, capillary, and other cardiovasculartissue, for example, heart chambers of other heart regions or heart orcardiac valves or valve structures), renal tissue (for example renalpyramids of the kidney), liver tissue, cartilaginous tissue (e.g. nasalor auricular), bone tissue, skin tissue, and any other tissue or organor portion thereof that is being engineered on a synthetic scaffold.

In some embodiments, the cells may be allowed to proliferate on thescaffold for a time period, in which the cells can grow to formcolonies, after which the colonies can fuse to form a network of cells,and subsequently forming a tissue. Generally, the time for proliferationcan range from a few hours or days to a few weeks, such as about 1 dayto about 4 weeks, or about 1 day to about 2 weeks, or about 1 day toabout 1 week, or about 1 day to about 4 days. The time for proliferationcan also depend on the cultivation conditions for the cells. Parametersof the cultivation condition can include, for example, temperature, pH,amount of water, pressure, nutrients present, and type of cell.Cultivation conditions of cells are known in the art and can thereforebe adapted by a person skilled in the art depending on the desired celltype and application.

The term “tissue” refers to a structure formed by related cells joinedtogether, wherein the cells work together to accomplish specificfunctions. An organ refers to a differentiated structure of an organismcomposed of various cells or tissues and adapted for a specificfunction. Therefore, one or more species of living cells can be addedinto the mixture to form a specific organ. For a non-limiting example,the heart which is an organ contains muscle tissue that contracts topump blood, fibrous tissue that makes up the heart valves and specialcells that maintain the rate and rhythm of heartbeats.

As used herein, the term “seeding” refers to plating, placing and/ordropping the cells of the present invention into the electrospunscaffold of the present invention. It will be appreciated that theconcentration of cells which are seeded on or within the electrospunscaffold depends on the type of cells used and the composition of thescaffold.

Electrospun Scaffold

In some embodiments, at least a portion of the scaffold is produced byelectrospinning. In some embodiments, portions produced byelectrospinning may be connected such as by epoxy glue.

As used herein, the term “electrospinning” refers to a technology whichproduces electrospun fibers (e.g. nanofibers) from a polymer solution.During this process, one or more polymers are liquefied (i.e. melted ordissolved) and placed in a dispenser. An electrostatic field is employedto generate a positively charged jet from the dispenser to thecollector. Thus, a dispenser (e.g., a syringe with metallic needle) istypically connected to a source of high voltage, preferably of positivepolarity, while the collector is grounded, thus forming an electrostaticfield between the dispenser and the collector. Alternatively, thedispenser can be grounded while the collector is connected to a sourceof high voltage, preferably with negative polarity. As will beappreciated by one ordinarily skilled in the art, any of the aboveconfigurations establishes motion of positively charged jet from thedispenser to the collector. Reverse polarity for establishing motions ofa negatively charged jet from the dispenser to the collector is alsocontemplated. At the critical voltage, the charge repulsion begins toovercome the surface tension of the liquid drop. The charged jets departfrom the dispenser and travel within the electrostatic field towards thecollector. Moving with high velocity in the inter-electrode space, thejet stretches and the solvent therein evaporates, thus forming fiberswhich are collected on the collector forming the electrospun scaffold.

In some embodiments, the inner diameter and wall thickness of the tubesare adjusted by changing the collecting mandrels and controlling thedeposition time of electrospinning, respectively. In some embodiments,the porosity of a porous tube correlates with fiber diameter and polymerweight concentration, which enable manipulation of the scaffold porosityby changing the polymer weight concentration. In some embodiments, aporous scaffold produced by electrospinning exhibits permeability withinthe permeability range for human trabecular bone as exemplified below(permeability constant K=10-10-10-12 [m2]). In some embodiments, Celladherence is supported by the high surface area-to-volume ratios of theelectrospun nanofibers, whose nanoscale architectures expose the cellsto more binding sites compared with micro- and macro-scale architecture,and by that lead to a better adherence of every cell by allowing itsattachment to multiple nanofibers.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

In the discussion unless otherwise stated, adjectives such as“substantially” and “about” modifying a condition or relationshipcharacteristic of a feature or features of an embodiment of theinvention, are understood to mean that the condition or characteristicis defined to within tolerances that are acceptable for operation of theembodiment for an application for which it is intended. Unless otherwiseindicated, the word “or” in the specification and claims is consideredto be the inclusive “or” rather than the exclusive or, and indicates atleast one of, or any combination of items it conjoins.

It should be understood that the terms “a” and “an” as used above andelsewhere herein refer to “one or more” of the enumerated components. Itwill be clear to one of ordinary skill in the art that the use of thesingular includes the plural unless specifically stated otherwise.Therefore, the terms “a,” “an” and “at least one” are usedinterchangeably in this application.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

In the description and claims of the present application, each of theverbs, “comprise,” “include” and “have” and conjugates thereof, are usedto indicate that the object or objects of the verb are not necessarily acomplete listing of components, elements or parts of the subject orsubjects of the verb. Other terms as used herein are meant to be definedby their well-known meanings in the art.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated herein above and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, CT (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Materials and Methods Electrospinning of Fibrous Tubes

A 9% solution of polycaprolactone (PCL, Mw 80,000 Da; Sigma-Aldrich) ina 80:20 (w/w) mixture of chloroform (Frutarom) and dimethylformamide(DMF, Frutarom) was electrospun using the following parameters: anapplied voltage of 12 kV, a flow rate of 2.5 ml/hour and a tip-collectordistance of 12-17 cm. PCL fibrous tubes were collected using a rotatingaluminum wire with a diameter of either 0.5 mm or 2.8 mm, at 350 rpm.The collected tubes were then dried in a vacuum, at a pressure of ˜10-3atm, and then stored in a desiccator at relative humidity of ˜30%.

Capillary Design

Tubular scaffolds with an inner diameter of 2 mm, wall thickness of 0.2mm±0.02 mm and a length of 25 mm, were successfully constructed (FIG. 2a). Inner diameter and wall thickness of electrospun tubular scaffoldscan be adjusted by using mandrels with different external diameters andby controlling the electrospinning deposition time, respectively. Thedesign may be adjusted according to a desired application. SEM images ofthe electrospun scaffolds are shown in FIGS. 2b -d. All scaffolds showedrandomly oriented fibers and interconnected pore structure throughoutthe scaffold. However, fiber diameter and pore size were distinct. Toquantify the permeability of the scaffolds, different air pressure dropthrough 10×10×0.1 mm³ layers of scaffolds was applied and the totalpressure drop (dP) was measured using a commercially available watermanometer. Permeability was calculated using Equation 1.

$\begin{matrix}{v = {{- \frac{k}{\mu_{a}}} \cdot \frac{dP}{dx}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where ν is the flux (discharge per unit area, with units of length pertime[m/s]), dP/dx is the pressure gradient vector (Pa/m), and μ_(a) isthe air viscosity (Pa·s).

Fabrication of the Complex Scaffold

The complex scaffold is composed of a vasculature-like system embeddedin a bulk scaffold. The vasculature-like system is comprised ofelectrospun PCL tubes, whereas the bulk surrounding scaffold includesbone-inducing material (Pro-Osteon particles of 0.5-1 mm diameter,Bepex) and electrospun PCL fibers (FIGS. 4 & 5 a). Using a wet brush,the Pro-Osteon particles are dispersed in between and on the PCL tubesand are kept in place by the PCL fibers that also are dispersed aroundthe tubes and Pro-Osteon (FIGS. 4a & 5 b). Each vasculature structureconsists of three or more small electrospun PCL tubes (inner diameter.0.5 mm, FIG. 5c ), connected together from both sides by anotherelectrospun PCL tube with a diameter of 2.8 mm (FIG. 4b ). The lattertubes resemble inlet and outlet grafts for media infusion into theconstruct. The small tubes tips are inserted into the large one andconnected to it using an epoxy adhesive that forms a liquid-tightadherence. The radius of the entire scaffold system is adjusted to ˜1cm. All experiments were performed 3 or 4 times at least, and more, ifnecessary. All data are expressed as mean ±standard deviation (SD).

Example 1 Preliminary Capillary Design

As mentioned in the method section, inner diameter and wall thickness ofelectrospun tubular scaffolds can be adjusted. The average fiberdiameter and pore size increased as a function of the PCL weightconcentration in the electrospun polymer solution. More specifically,the mean diameter of fibers electrospun from the low concentration (8wt. %) PCL solution was 200 nm±65 nm, while for the higher PCL solutionconcentration (11 wt. %) mean fiber diameter was 550 nm±45 nm. Asexpected, the porosity of the electrospun scaffold is correlated withthe fiber diameter, namely, higher porosity was observed in scaffoldswith fibers of larger diameter. Additionally, the permeability of thescaffolds was quantified using Darcy equation, and the Darcy constant kwas found to be in the range of 10⁽⁻¹⁰⁾-10⁽⁻¹²⁾ [m²].

Example 2 Preliminary Flow and Permeability Study Using HomemadeBioreactor

Flow and tube wall permeability were characterized by tube assembly in ahomemade bioreactor. The tube was connected to an inlet and outlet (FIG.3) and immersed in a PBS bath (Invitrogen). A 10% solution of fetal calfserum (FCS) serum in PBS (Invitrogen) was pushed through the tube usinga peristaltic pump, while the longitudinal pressure was regulated, inthe range of 1-2 N/m2, with a valve close to the outlet. The serum thatpassed through the PCL wall tubes (the permeate) entered the surroundingbath and the excess fluid (concentrate) exited and collected in the feedreservoir (FIG. 3). The permeate was collected during the experiment andprotein concentration was measured using a native 10% gelelectrophoresis system.

The PCL tubes proved permeable, as demonstrated from the increasingprotein concentrations measured over time in the PBS bath in whichperfused tubes were immersed (FIGS. 7A-C). The system reached saturationwithin 120 minutes of perfusion. The tubes were permeable to all proteinsizes present in the serum.

Example 3 Preliminary Adjustment of Culture Conditions

Human adipose-derived mesenchymal stem cells (MSCs) were isolated fromhuman adipose tissue explants (1-2 mm³) extracted from abdominal area.This study was approved by the Rambam Health Care Campus HelsinkiCommittee (#0370-12-RMB). The explants were placed in fibronectin-coatedflasks and incubated in a basic growth medium, containing Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum(FBS), 1% L-glutamine and 1% Pen-strep antibiotics (all from BiologicalIndustries) for 5-7 days. After one week, adipose tissue explants werewashed and MSCs were trypsinized. MSCs were cultured in a basic growthmedium, which was changed once in 3 days. Cells were expanded up topassage 3 or 4 in growth medium and were then used in in vitro and invivo studies.

Inductive conditions were achieved by culturing MSCs in inductive mediumcomprised of DMEM, supplemented with 10% FBS, 10⁻⁸ M dexamethasone and100 μg/ml L-ascorbic acid 2-phosphate sesquimagnesium (both from SigmaAldrich). Moreover, osteogenic differentiation was induced by culturingthe cell-seeded scaffolds in osteoconductive medium comprised ofinductive medium further supplemented with 10 mM β-glycerophosphate(Sigma Aldrich).

MSCs were only seeded onto scaffold regions containing Pro-Osteons (FIG.2a ). The seeding region dimensions were approximately 2.2 cm long and 1cm wide. Prior to seeding, all scaffold samples were soaked in 70%ethanol for sterilization and washed several times with phosphatebuffered saline (PBS). One million of trypsin-released cells werecounted and re-suspended in 50 μl growth or inductive medium and wereseeded onto the scaffold. The cell-seeded samples were then incubatedfor 70 min with slow rotation, and then re-suspended in growth orinductive medium, respectively.

Proliferation of MSCs cells in a static culture: To evaluate theproliferation rates and growth viability of the isolated MSCs, theAlamar blue-based viability assay was used. Cells were seeded on thescaffolds and cultured in growth medium for 28 days. Cell viability wasassessed on days 1, 3, 7, 14, 21 and 28 post-seeding. At each timepoint, the cell-seeded scaffold was washed twice in PBS and incubatedfor 2 hours in medium containing 10% Alamar blue reagent (Serotec, UK).The fluorescence of Alamar blue reagent was recorded by FLUOstar galaxyfluorescence reader (BMG Labtech, Germany) at 540 nm excitation and 580nm emission.

Histological analysis of cell-seeded scaffolds cultured under dynamicconditions: MSCs were seeded on the complex scaffolds (˜1 million cellsper scaffold) and cultured for two weeks in a medium flow bioreactorsystem (FIG. 6). During the first week, the cells were cultured ininductive medium, before being transferred to osteoconductiveconditions. For histological staining, cell-seeded scaffolds were fixedin 10% neutral buffered formalin (NBF) for 48 hours, and thendecalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution for 1week. The constructs were then embedded in paraffin after undergoingstandard fixation. Transverse, 5 μm-thick sections were placed onsilanized slides for hematoxylin & eosin (H&E) or Masson's Trichromestaining.

Morphological analysis of cell-seeded complex scaffolds cultured underdynamic conditions: MSCs were seeded on complex scaffolds, which werethen cultured in growth medium for approximately 1 month in a mediumflow bioreactor system. Samples were then fixed in 10% NBF, dehydratedin graded ethanol solutions and soaked in hexamethyldisilazane for 15minutes. The samples were then sputter-coated with gold andcharacterized using a Phenom desktop scanning electron microscope (SEM)(5 kV accelerating voltage, FEI Company).

Static Culture Conditions

The metabolic activity rate and proliferation capacities of MSCscultured on complex scaffolds under static conditions increased indirect correlation with the duration of culture, as presented in FIG. 8.This result is a preliminary indication of the effective biologicalsupport provided by the complex scaffold.

Dynamic Culture Conditions

After examining the biocompatibility of the scaffold in a staticculture, it was tested in a dynamic culture. An MSC-embedded complexscaffold was first cultured for one week under static conditions andthen transferred for an additional week in an inductive medium,delivered via a medium flow bioreactor system (FIG. 6). The scaffold wasthen fixed, decalcified, embedded in paraffin and stained. TheH&E-stained histological sections (FIGS. 9A-D of the seeded scaffold,illustrate the complex structure of the scaffold that consisted of threePCL tubes embedded within a bulk tissue (asterisks indicate the tubes).The images also demonstrated the extensive MSC proliferation and theexpansion within the scaffold. It was also noted that MSCs did notpenetrate the PCL tubes, but rather, settled on the wall of the tubes.

The SEM analysis corroborated these findings (FIGS. 10A-C), as well asexcellent integration of the cells within the scaffold. Cells weredistributed along and in between the osteoconductive particles,depositing their extracellular matrix components throughout the bulkpart of the scaffold. In addition, cells were observed along theexternal walls of the embedded tubes. It is important to state that incontrast to histological sections, wherein the ceramics are dissolvedthrough a decalcification process, the Pro-Osteon particles can still beseen in the SEM analysis.

Example 4 Preliminary Implantation of MSC-Seeded Scaffolds in EctopicModels

In order to examine the biocompatibility of the complex scaffolds invivo, they were seeded with induced cells and then implanted in ectopicanimal models.

On the implantation day, complex scaffolds were seeded with cells thatwere previously cultured for at least 10 days in inductive medium. Priorto implantation, a fibrin clot, composed of 1:1 rat fibrinogen: ratthrombin (Sigma Aldrich), was added to the cell-seeded constructs tostabilize the sample.

All surgical processes described below were performed following theprotocols approved by the Institutional Animal Care and Use Committee.Three groups of 6-week-old, nude female mice (n=5 per group, HarlanLaboratories) were anesthetized using a 0.5:0.5:9 ketamine:xylazine:PBScocktail at a dose of 400 μL/20 g, delivered with a 25-gauge needle.Cell-seeded complex constructs were subcutaneously implanted to thedorsum of the anesthetized mice. In parallel, unseeded scaffolds andseeded Pro-Osteons particles were subcutaneously implanted as negativeand positive controls, respectively. Tissue samples of the constructarea were extracted for histological analysis 8 weeks post-implantation.

Histological sections of the implanted scaffolds extracted 8 weeks afterimplantation, showed the new formed tissues around the Pro-Osteons(crosses represent their position before decalcification; FIGS. 11C, Eand H) and the vasculature-like system within the implanted scaffold,represented by three PCL electrospun-tubes (asterisks; FIGS. 11A, F andH). Moreover, the PCL fibers that constitute the tubes wall (blackarrows; FIGS. 11D and I), MSC integration within the scaffold (blackdots examining the cells' nuclei), formation of blood vessels (redarrows; FIGS. 11B, F, G and H) and muscle tissue (yellow arrows; FIGS.11A and F) and accumulation of collagen into the scaffold (blue arrows;FIGS. 11B, F and G) were observed. Furthermore, maintained structuralintegrity of vasculature-like scaffold was apparent and no inflammatoryreaction toward the graft was detected, indicating its biocompatibilitywith the host.

Example 5 A Custom Designed Hollow Fiber Reactor (HFR) System forDynamic Culturing

A hollow fiber reactor (HFR) (1270) was developed for use with themicrocapillary system to enable cell growth and differentiation prior totransplantation. The bioreactor system (FIGS. 12A and 12B) is based on500 ml Erlenmeyer flask used as a medium reservoir (1271); it has aspecially designed cap (1272) that contains inlets and outlets for feed,waste and aeration. The microcapillary system (1273) is placed into theHFR vessel (1274), connected to an inlet tube (1275) and an outlet tube(1276) and then the growth medium is constantly circulated throughoutthe system. Cell seeding is conducted using the HFR inoculation port(1278). Following cell seeding onto microcapillary system (1273), it isclosed and during all of the 3D growth phase maintains closed systemparameters.

Seeding conditions: 8-12×10⁶ MSCs in 300 ul growth medium were injectedonto the construct (using the inoculation port of the HFR) in severalpoints on the construct and incubated at static conditions for adequatecell adherence. Following 20 minutes, 2 ml growth medium was added andincubation continued at static conditions (37° C., 5% CO2) for total of120 minutes. After the incubation, the HFR vessel was connected to thesystem tubing and the growth medium (total volume of 150 ml) circulationthrough the microcapillary construct began (at 18 RPM based onpreliminary studies). The circulating medium was aerated by air+5% CO2mix into the medium reservoir using a sparger (aeration rate is 15ml/hour). Medium change (50 ml) was performed twice a week using thewaste and the feed ports on the cap. The bioreactor system was verystable and was able to run during a long period (one month wassuccessfully tested). Osteogenic induction was performed for two days byadding BMP2 (150 ng/ml final conc.) to the growth medium.

Example 6 Cells Characterization Following Culturing of MSCs on theMicrocapillary Scaffold in the HFR Dynamic System Compared to a StaticSystem a. Cell Attachment and Growth on the Microcapillary Scaffold

Since cell counting on the microcapillary scaffold is challenging, cellsgrowth and construct coverage is shown using Giemsa staining, whichdemonstrates the MSCs growth onto the microcapillary system (FIGS. 13Aand B, magnification ×1).

Histological evaluation (Hematoxylin & Eosin staining) after one week ofculturing in the HFR system supports MSCs growth onto the scaffoldsconstruct. As can be seen in the images (FIGS. 14A and B), the cellsbetween the mineral particles, embedded within the PCL fibers, havegenerated organized connective tissue around and between the mineralparticles.

Cells viability was demonstrated by MTT staining (FIGS. 15A and B). Thelive cells are violet colored (15A) and the control mineral particles(without cells) are not colored (15B). This method demonstrates cellscoverage as well as viability.

b. Osteogenic Potential of MSCs Following Osteogenic Induction by BMP-2,in Static and Dynamic Growth Systems

In order to evaluate the MSCs osteogenic potential following cultivationin the HFR system experiments ending with osteogenic induction of thecultured cells were performed. The experiments compared static anddynamic (HFR) growth systems. The static system is composed of a petridish or in an Erlenmeyer in which cells are cultured on themicrocapillary scaffold and medium is manually replaced. The dynamicsystem is composed of a fully closed bioreactor which enables mediumaeration and flow along and within the microcapillary graft, as well asan automatic medium replacement by specific ports.

Osteogenic induction was evaluated by the osteogenic marker ALP, usingFACS (FIGS. 16 A-B). Results show that cells cultivated in the dynamicHFR system had a higher level of ALP positive cells (33.23%, 16B)compared to the level of ALP positive cells (18.65%) of cells cultivatedin the static system (16A).

Osteogenic differentiation was also evaluated using Real-Time PCR (FIGS.17A-C). The RNA was extracted using the PureLink® RNA Mini Kit (Lifetechnologies). TaqMan osteogenic primers (DLX5 and Osterix (SP7)) wereused for Real-Time reactions preparation. The osteogenic genesexpression, following 2 days of osteogenic induction (day 9 ofcultivation), was compared to the osteogenic genes expression at HFRseeding day (day 0). Both dynamic and static growth conditions wereevaluated. Real time PCR results demonstrated DLX5 and SP7 geneexpression are elevated post osteogenic induction (day 9) relative today 0, indicating for osteogenic induction in both systems, with higherexpression in dynamic HFR system compared to static system.

c. Endothelial Cells (ECs) Growth on the Microcapillary Scaffold inDynamic and Static Culturing Systems

The ability of the microcapillary system to support Seeding and growthof Human Adipose Microvascular Endothelial Cells (HAMEC) inside themicrocapillary tubes was tested as following: the growth system wasbuilt as was described before, but with single PCL tube. Before cellseeding, the tube was coated with fibronectin to allow adherence ofendothelial cells onto the inner surface of the tube lumen. The sterilePCL tube was aseptically filled with fibronectin solution and rotatedfor one hour at 37° C. incubator to allow even coating with fibronectin.Subsequently, suspension of endothelial cells was prepared inEndothelial Cell Medium (ECM) (from ScienCell). To seed cells, the PCLtube was drained out of the fibronectin solution, and filled insteadwith the prepared suspension of endothelial cells. Cells were seeded ata density of 5×10⁵ cells/cm², and the tube was filled in the appropriatevolume of medium. ECM medium was also used to submerge the PCL tube tocover the outside surfaces and the tube was rotated for two hours at 37°C. incubator to allow even seeding of cells onto the inner surface ofthe tube. After two hour-cell-seeding, the PCL tube was opened from bothsides and was connected to the dynamic system including peristaltic pumpand aerated growth medium reservoir. The growth medium was circulatedconstantly as previously described. The medium flow was kept for 48hours. Next, the tube was taken out of the incubator and washed twicewith PBS and stained with Giemsa and visualized (FIG. 18A). Adequatecell coverage of the inner walls of the tubes is seen withcharacteristic cobble stone morphology. Moreover, the HAMECs retainedtheir AC-LDL uptake capabilities (FIG. 18B) which points out that theirbiological functionality is intact.

Example 7 Demonstration of Human Plasma Protein Permeability via theMicrocapillary Scaffold, in Conditions That Mimic Blood Flow Within theScaffold

The microcapillary construct permeability to human plasma proteins wastested using the system illustrated in FIGS. 19A-B. As demonstrated inthe schematic illustration of FIG. 19B, a sample of human plasma(diluted 1:4) 1991 was streamed into the microcapillary system 1992 in aHFR vessel 1993 using a peristaltic pump 1994 in a circular manner. Themicrocapillary system wetted as planned and liquid droplets formed onthe construct outer perimeter, the droplets were gathered using acollection tube 1995. Sampling the liquid gathered inside the collectiontube was sampled every 30 min for 5 hr. Human serum albumin, IgG andlysozyme concentrations were determined in every sample (using IMPLEN'sNanoPhotometer® P-Class) and were compared to the concentrations in theplasma before the experiment. Results demonstrate that the system ispermeable to all tested proteins: human serum albumin (FIG. 20A), IgG(FIG. 20B) and Lysozyme (FIG. 20C). Further a constant wetting rate wasobserved meaning there is no plugging of the system with time.

Example 8 Standardization and Stability of the Microcapillary Scaffold

The electrospinning machine includes syringe that contains the polymersolution which is 12% polycaprolactone (PCL, Mw 80,000 Da) in an 80:20(w/w) mixture of chloroform and dimethylformamide (DMF). The polymersyringe (5 ml BD plastic syringe) is driven by a syringe pump which isused to control the flow rate (1 ml/hour) of the polymer being ejected.The electrospinning machine also includes high voltage power supplywhich apply a fixed voltage of 15-20 kV to a metallic needle (21 G)connected to the polymer syringe. The polymer solution (12% PCL) passthrough the needle and is charged by the high voltage that is directlyopposite to the surface tension of the polymer solution, leading to theelongation of the hemispherical surface of the solution at the tip ofthe syringe to form a conical shape known as “Taylor cone”. Due toelongation and solvent evaporation, the charged jet forms randomlyoriented nanofibers that are collected on a grounded metallic collectormade of wire of 0.5 mm diameter, or tubes of 0.86 mm or 1.5 mm diameter.

Tubes of 0.5 mm diameter are the capillary-like tube and tubes of 0.86mm and 1.5 mm diameter are the vein-like tube that is cut to two tubesand connected to both sides of the capillary-like tube.

The vasculature-like system for in vitro experiments is made of threePCL tubes of 0.5 mm (collection time 8 minutes, flow rate 1 ml/hour)which are connected together from both sides by PCL tubes of 1.5mm(collection time 40 minutes, flow rate 1 ml/hour), which resemble aninlet and outlet grafts for media infusion into the construct. The threetubes are surrounded with pro-osteon particles that are dispersed inbetween and onto the PCL tubes and are kept in place by the PCL fibers.

Example 9 Demonstration of Microsurgery Technique Proof of Concept—forTransplantation of the Microcapillary Scaffold End to End in a Rat Model

To provide compatibility between the diameters of the vein-like tube(inlet/outlet) and the rat femoral/artery vein, a vein-like tube havinga diameter of 0.86 mm was produced, using a collector of 0.86 mm by theelectrospinning technique. The adjustment in the vein diameter was doneto overcome any incompatibility between the diameters of the vein-liketube (inlet/outlet) and the rat femoral/artery vein that can lead toblood leakage from the anastomosis site the vein-like tube diameter. Inaddition, the number of capillary-like tubes connected to the vein-liketubes was adjusted to two capillary-like PCL tubes of 0.5 mm. These twocapillaries were surrounded by bone-inducing material (pro-osteon) andelectrospun PCL fibers, and connected together to 0.86 mm vein-like tubein both sides using PCL solution as glue.

Next, the system was sterilized and transplanted in Sprague Dawley (SD)rat model using microsurgery technique. The vasculature-like system wasfixated to the rat muscle tissue, then anastomosed to the femoral arteryin one side and the femoral vein in the other one by 6-8 stitches ineach side done with 10-0 Prolene microsurgical suture via “End to End”anastomosis (FIG. 21A-H). After finishing the anastomosis and releasingthe clamps that held the artery and vein, no blood was leaked from theconnection site. Using laser Doppler, the blood flow in the artery andvein was measured (FIG. 22A), demonstrating a blood flow through thevasculature-like system (FIG. 23). Later, the rat skin was stitchedabove the transplant and coated with polydine ointment to prevent anyinfections. After the surgery, the rat was examined for a week; and nopain was shown by the rat and it moved freely which mean that no damagewas done to its limbs although it lost the main blood supply.

One day later, the rat was anesthetized and the stitches were cut of theskin in order to measure the blood flow through the femoral artery andvein. As demonstrated in FIGS. 23A-B the blood still flowed through theanastomosed vasculature-like system but it was reduced by 41-47%according to the laser Doppler (FIGS. 23C and 23D), probably as a resultof blood clots. Two weeks later, the rat was sacrificed and thetransplant was extracted (FIG. 24).

Next, the extracted transplants were fixated, Paraffin-embedded blockswere prepared and stained with Hematoxylin & Eosin. Histologicalanalysis of the transplant showed that the scaffold structure ismaintained, the fibers of PCL tubes were not destroyed and there isaccumulation of cells into the capillary-like tubes (FIG. 25A-D).

In order to prevent clotting inside the vasculature-like system duringthe surgery, the scaffold was soaked with phosphate buffered Saline(PBS), and 2 ml of Heparin were injected (5000 unit/ml) through it to besoaked with heparin. Next, the heparin-soaked scaffold was anastomosedto the femoral artery from one side, blood passage through the scaffoldwas examined by releasing the blocking clamp for seconds (FIG. 26A).Next, the other side was connected to the femoral vein. After releasingthe clamps, the blood flowed through the scaffold without any leakagefrom the connection site (FIG. 26B). In addition, every rat was injectedsubcutaneously with 75 Units/kg of heparin after completing the surgery.Three weeks later, the rats were anesthetized; their blood flow throughthe transplants was measured (FIG. 26C), and the laser Doppler showedthat although much improved, there is still a slight reduction in theblood flow (FIG. 26D).

In the following surgeries, in order to overcome any clotting that mayoccur in the transplant and reduce the blood flow through it; a dailydosage of 100 units/kg heparin was inject for a week after thetransplantation. There was no bleeding as a result of heparin and therats acted and moved normally.

Example 10 Calibrated Flow Rates

The flow rates were evaluated using a calibrated peristaltic pump andrelevant blood flow rates obtained from the Literature. The pressure onthe blood vessel wall is influence by the blood flow rate inside theblood vessels and this pressure in turn is the driving force for bloodpenetration throws the walls. As mention above, in the large bloodvessels it is not desirable process, while in capillary it is necessaryprocess for the cell nourish. Since the blood flow rate influence by theblood velocity and the cross section of the blood vessels, penetrationtests should be evaluated in the relevant flow rates.

The peristaltic pump was calibrated in order to examine the range of itsflow rate. Two pump rate were examined (high 100 rpm and low 11 rpm) andthe coming out fluid volume was measured after defined time (20-75minutes). Flow rate (Q) was calculated using Equation 2 (V—volume,t—time) and the flow velocity was measured using Equation 3 (v—velocity,A—area=πr²).

$\begin{matrix}{{Q\left( \frac{ml}{Sec} \right)} = \frac{V({ml})}{t\left( \sec \right)}} & {{Equation}\mspace{14mu} 2} \\{{v\left( \frac{cm}{Sec} \right)} = \frac{Q\left( {{ml}/\sec} \right)}{A\left( {cm}^{2} \right)}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

The results are shown in Table 1, while blood flow velocity in differenttypes of blood vessels is demonstrated in Table 2. FIG. 8 shows the flowrate of the peristaltic pump in correlation with pump rate.

TABLE 1 Peristaltic pump calibration and flow rate calculation Volume(ml) Time (min) Flow Rate (ml/hr) Pump Rate (90/10) = 100 rpm 21 2063.00 48 40 72.00 62 72 51.67 Q (ml/hr) Mean 62 SD 10 Q (ml/sec) 0.017 v(cm/sec) 0.152 Pump Rate (10/1) = 11 rpm  5 20 15.00  8 48 10.00 11  63.5 10.39 Q (ml/hr) Mean 12 SD 3 Q (ml/sec) 0.003 v (cm/sec) 0.029

TABLE 2 Peristaltic pump calibration and flow rate calculation Relationbetween blood flow velocity and total cross-section area in human ^([1])Blood cross- Blood velocity Vessel section flow Type of in cm/s diameterarea rate blood vessels V (cm/s) D (cm) A (cm²) Q (cm³/sec) Aorta 40 2.54.91 196 Capillary 0.03^([2]) 0.0008 0.000001 0.00000002 Vein 15 0.50.20 3 Tube like Vain 0.152 0.38 0.11 0.0172

The fibers penetration levels of our tubes like vain and capillaryfibers were evaluated based on fiber diameter, fiber thickness—dependingon electrospinning duration—and PBS flow rate. Electrospun PCL tubes atdifferent diameters and thickness were connected to the bioreactorsystem in order to examine their permeability (FIG. 27). The tubepermeability was calculated using Equation 4.

$\begin{matrix}{{\%\mspace{14mu}{permeability}} = {{\frac{{leakage}\mspace{14mu}{rate}\mspace{14mu}\left( {{ml}/\sec} \right)}{{Pump}\mspace{14mu}{flow}\mspace{14mu}{rate}\mspace{14mu}\left( {{ml}/\sec} \right)} \cdot 100}\%}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

As demonstrated in the results below, tubes simulating veins were almostnot penetrable. However, tubes simulating capillaries were 65%permeable. Moreover, it seems that ˜40 minutes of electrospinning issufficient to block almost completely the fiber wall. (FIG. 28).

What is claimed is:
 1. A method of producing a tissue, the methodcomprising: providing a scaffold comprising: at least one inlet tube; atleast one outlet tube; a plurality of porous elongated microtubes,wherein each one of said porous elongated microtube has an innerdiameter of 5-100 micrometers, wherein said plurality of elongatedmicrotubes extend from said at least one inlet tube to said at least oneoutlet tube and is in fluid communication thereto; and a plurality offibers having a diameter range of 0.5-10 micrometers, wherein saidplurality of fibers is dispersed upon a portion of each of saidplurality of porous elongated microtubes; seeding cells on saidplurality of porous elongated microtubes of said scaffold; and providingliquid containing nutrients through said inlet of said scaffold, so asto provide nutrients from pores of said plurality of porous elongatedmicrotubes to said cells; thereby producing said tissue.
 2. The methodof claim 1, wherein cells are seeded on and/or within said plurality offibers.
 3. The method of claim 1, wherein said tissue is suitable forbeing implanted into a subject in need thereof.
 4. The method of claim1, wherein said inlet and said outlet of said scaffold is suitable forbeing surgically connected to a vascular system of a subject in needthereof, thereby providing fluid communication between the subject'svascular system and said scaffold.
 5. The method of claim 1, whereinsaid cells are selected from the group consisting of: adipose-derivedstem cells, mesenchymal cells, mesenchymal stem cells, vascular smoothmuscle cells, adipogenic cells, osteoprogenitors cells, osteoblasts,osteocytes, chondroblasts, chondrocytes and osteoclasts, endothelialprogenitor cells, hematopoietic progenitor cells, micro vascularendothelial cells and macro vascular endothelial cells, beta cells,hepatocytes and a combination thereof.
 6. The method of claim 1, whereinsaid scaffold further comprising plurality of bioactive particlesembedded in between said plurality of fibers.
 7. The method of claim 6,wherein said bioactive particles have a range of 200-1500 micrometers indiameter.
 8. The method of claim 6, wherein said plurality of bioactiveparticles are one or more type of osteoconductive particles.
 9. Themethod of claim 6, wherein the one or more types of the osteoconductiveparticles are selected from the group consisting of: calcium carbonate,hydroxyapatite (HA), demineralized bone material, morselized bone graft,cortical cancellous allograft, cortical cancellous autograft, corticalcancellous xenograft, tricalcium phosphate, corraline mineral andcalcium sulfate.
 10. The method of claim 1, wherein a portion of saidscaffold is printed, molded, casted, polymerized, or electrospun. 11.The method of claim 1, wherein at least one of said inlet tube, saidoutlet tube and said porous elongated microtubes are electrospun tubes.12. The method of claim 11, wherein said electrospun tubes comprise apolymer selected from the group consisting of: biodegradable polymers,non-biodegradable polymers and a combination thereof.
 13. The method ofclaim 12, wherein said polymer is selected from the group consisting of:polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA),and poly(Lactide-co-Glycolide) (PLGA), poly(orthoester), apoly(phosphazene), poly(or polycaprolactone, polyamide, polysaccharide,albumine and collagen.
 14. The method of claim 1, wherein said inlettube and said outlet tube have a wall thickness range of 50-2,000micrometers.
 15. The method of claim 1, wherein said plurality of porouselongated microtubes has a wall thickness range of 0.5-50 micrometers.16. The method of claim 1, wherein said inlet tube and said outlet tubehave an inner diameter of range of 2,000-10,000 micrometers.
 17. Themethod of claim 1, wherein an average diameter of a pore of saidplurality of porous elongated microtubes is 0.1-5 micrometers.
 18. Themethod of claim 1, further comprising providing the scaffold at leastone agent for promoting cell adhesion, colonization, proliferationand/or differentiation.
 19. The method of claim 18, wherein the at leastone agent for promoting cell adhesion is selected from the groupconsisting of: gelatin, fibrin, fibronectin and collagen.